Na(+)/H(+) exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na(+)/H(+) exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1-355), or a truncated mNHERF(1-325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) at 10(-4) M inhibited Na(+)/H(+) exchange activity in control and OK cells expressing wild-type mNHERF(1-355) by >60% but by <10% in cells expressing mNHERF(1-325). NHE3 coimmunoprecipitated with mNHERF(1-325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1-325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3-O-methyl-D-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.